Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The present invention provides a method, upon introducing an extracellular
substance, such as sperm, a cell nucleus, a nucleic acid, and a protein,
into a mammalian oocyte, for introducing the extracellular substance into
a mammalian oocyte, which is safe for the mammalian oocyte, enables
efficient introduction, and is technically easy. By treating the
mammalian oocyte with a tubulin-polymerization inhibitor such as
vinblastine, membrane fusion between the oocyte and the sperm is
promoted, and the sperm and the oocyte are fused without making a hole in
the oolemma and fertilization can be achieved. The method of the present
invention can be applied not only to the case of introducing sperm into a
mammalian oocyte, but also to the case of introducing an extracellular
substance, such as a cell nucleus, a nucleic acid, and a protein, into a
mammalian oocyte.

Claims:

1. A promoter for introducing an extracellular substance for a mammalian
oocyte used for introducing an extracellular substance containing a
tubulin-polymerization inhibitor as an active ingredient into a mammalian
oocyte.

2. The promoter for introducing an extracellular substance for a mammalian
oocyte according to claim 1, wherein the tubulin-polymerization inhibitor
is vinblastine.

3. The promoter for introducing an extracellular substance for a mammalian
oocyte according to claim 1 or 2, wherein the introduction of an
extracellular substance into a mammalian oocyte is the introduction of a
sperm into the mammalian oocyte.

4. The promoter for introducing an extracellular substance for a mammalian
oocyte according to claim 3, wherein the introduction of a sperm into the
mammalian oocyte is promoted by enhancement of fertility of the mammalian
oocyte.

5. The promoter for introducing an extracellular substance for a mammalian
oocyte according to any of claims 1 to 4, wherein the mammal is a human.

6. The promoter for introducing an extracellular substance for a mammalian
oocyte according to any of claims 1 to 4, wherein the mammal is selected
from the group consisting of a mouse, rat, cattle, pig, horse, sheep, and
monkey.

7. A method for introducing an extracellular substance into a mammalian
oocyte, wherein the mammalian oocyte is treated with a promoter for
introducing an extracellular substance for a mammalian oocyte containing
a tubulin-polymerization inhibitor as an active ingredient, when
introducing the extracellular substance into the mammalian oocyte.

8. The method for introducing an extracellular substance into a mammalian
oocyte according to claim 7, wherein the mammalian oocyte is treated with
vinblastine, when introducing the extracellular substance into the
mammalian oocyte.

9. The method for introducing an extracellular substance into a mammalian
oocyte according to claim 7 or 8, wherein the introduction of an
extracellular substance into a mammalian oocyte is the introduction of a
sperm into the mammalian oocyte.

10. The method for introducing an extracellular substance into a mammalian
oocyte according to claim 9, wherein the introduction of an extracellular
substance into a mammalian oocyte is achieved by sperm-oocyte fusion in
vitro.

11. The method for introducing an extracellular substance into a mammalian
oocyte according to claim 9 or 10, wherein the introduction of a sperm
into a mammalian oocyte is promoted by improvement of fertility of the
mammalian oocyte.

12. The method for introducing an extracellular substance into a mammalian
oocyte according to claim 11, wherein the improvement of the fertility of
the mammalian oocyte is promotion of sperm fusion to an oolemma.

Description:

TECHNICAL FIELD

[0001]The present invention relates to a promoter for introducing an
extracellular substance for a mammalian oocyte containing a
tubulin-polymerization inhibitor as an active ingredient, and to a method
for introducing an extracellular substance into a mammalian oocyte by
using the introducing promoter. More specifically, the present invention
relates to a method for introducing an extracellular substance such as a
sperm, cell nucleus, nucleic acid, and protein, into a mammalian oocyte
by using a promoter for introducing an extracellular substance for a
mammalian oocyte containing a tubulin-polymerization inhibitor as an
active ingredient, and to a promoter for introducing an extracellular
substance for a mammalian oocyte to be used therefor. One characteristic
of the invention is to provide a method for promoting fertilization of a
mammalian oocyte, by promoting introduction of a sperm into a mammalian
oocyte, by enhancing fertility of a mammalian oocyte by a treatment with
a promoter for introducing an extracellular substance for a mammalian
oocyte containing a tubulin-polymerization inhibitor as an active
ingredient.

BACKGROUND ART

[0002]In the field of mammalian modification using gene therapy,
reproductive treatment, or developmental engineering, the introduction of
an extracellular substance such as a sperm, cell nucleus, and nucleic
acid into a mammalian oocyte is conducted in order to modify germ cells.
As a method for introducing genetic (nucleic acid) substances into a cell
such as a mammalian oocyte, an introduction method using vectors such as
retroviruses is known hitherto (WO97/18318). As a method for introducing
an extracellular substance such as a sperm and cell nucleus into a
mammalian oocyte, the ICSI (intracytoplasmic sperm injection) method is
generally used, in which a hole is made in an oolemma using a glass
capillary, thereby infusing an extracellular substance. The ICSI method
is also a mainstream method for introducing a sperm into an oocyte of
mammalians including human (Japanese Laid-Open Patent Application No.
5-38345), and which is broadly used for treatment of human infertility.

[0003]However, the ICSI method has some problems as a method for
introducing an extracellular substance into a mammalian oocyte. More
specifically, the ICSI method has the following problems: (1) technical
training is required in order to introduce an extracellular substance
into a mammalian oocyte without fail by the ICSI method; (2) the success
rate of introducing an extracellular substance into a mammalian oocyte by
the ICSI method is low; (3) since the ICSI method is carried out by
making a hole in an oolemma using a glass capillary, an adverse effect on
embryonic development caused by making a hole can be conceivable. Even
though the problems have been pointed out, the ICSI method is in use
since there is no alternative method for the ICSI method currently.

[0004]On the other hand, introduction of a sperm into a mammalian oocyte
is conducted by fertilization, and several methods for promoting
introduction of a sperm into a mammalian oocyte are proposed. For
example, Published Japanese translation of PCT international publication
8-503705 discloses a method for treating mammalian male infertility by
administrating a fertility-enhancing thymosin polypeptide such as
thymosin α1 to promote fertilization. Japanese Laid-Open
Patent Application No. 9-28721 discloses a method for improving in vitro
fertility of cattle sperm, in which cattle sperm is cocultured with a
cattle oviduct epithelial cell, then cultured with a cattle oocyte to
undergo in vitro fertilization.

[0005]Furthermore, Published Japanese translation of PCT international
publication 2001-506225 discloses a method of using angiotensin II to
promote fertilization of a mammalian oocyte. Japanese Laid-Open Patent
Application No. 6-189650 discloses a method for increasing conception
rate by magnetizing a sperm and an oocyte and enhancing the activity
thereof. Also, Published Japanese translation of PCT international
publication 2001-500102 discloses a method for improving sperm-oocyte
binding and enhancing fertility by using a purified polypeptide purified
by centrifuging a frozen- and thawed-sperm suspension in the presence of
10% to 12% of glycerol, and being a member of a group of proteins
including prosaposin (SGP-1) and saposin. All of the methods are aiming
at obtaining an effect of activating an oocyte and sperm, and promoting
fertilization thereof during mammalian oocyte fertilization.

[0006]On the other hand, in a cell of eukaryotes such as animals, plants,
and fungi, it is known that there is an organelle called microtubules,
which is scattered in the cell and is tubular protein fibers having a
diameter of about 25 nm. The functions of organelles are extremely
wide-ranging and involved in mitotic apparatus formation, formation and
retention of cell morphology, flagellar/ciliary movement, placement of
intracellular organelles, material transportation, hormonal secretion,
and cytoplasmic membrane fluidity. Especially, microtubules are present
as the main constituent molecules of axons and dendrites in neurons, and
contribute to transportation of materials as rails for motor proteins.
Microtubules are generally formed by regular polymerization of a
heterodimer which consists of one α-tubulin molecule and one
β-tubulin molecule, and repeat expanding and contracting caused by
polymerization/depolymerization along with cell cycle progression.
Moreover, the polymerization/depolymerization of the microtubules is also
controlled by Microtubule-Associated Proteins (MAPs, τ protein), and
a protein kinase and protein phosphatase containing the
microtubule-binding proteins as a main substrate are involved in the
control mechanism of microtubules. From these facts, it is known that
compounds which act on the microtubule system show various vital
activities such as cell division inhibition.

[0007]As described above, microtubules are the main components of mitotic
spindle fibers which guide chromosomes arranged on the equatorial plate
at the time of mitosis to polar points, and play an indispensable roll in
many cellular actions including maintenance of the shape, motility, and
adhesion of cells, and transportation within cells, during division phase
(Shiff, P. B. and Horwitz, S. B., 1981, "Molecular Actions and Targets
for Anticancer Chemotherapeutic Agents" section of "Tubulin: a target for
chemotherapeutic agents", pp 483-507, Academic Press, NY, USA; Glotz, J.
S. et al., 1992, Proc. Natl. Acad. Sci., USA, 89:7026-7030; Rowinsky, E.
K. et al., 1990, Nature, 82:1247-1259). Also, microtubules play an
important role in the interactions between growth factors and receptors
on the surface of cells, and in controlling membrane-permeable
proliferative signals derived from these interactions. Relationship
between fertilization and cytoskeleton has been also reported (Molecular
Reproduction and Development, 56:89-98, 2000).

[0008]Tubulin is the main component protein of microtubules and an
indispensable protein for cell division/cell growth, which is a so called
cytoskeletal element. Therefore, polymerization inhibition or
depolymerization of tubulins has an important relationship with cell
division/cell growth. From such functions of tubulin, tubulin is also
known as a target of an anticancer agent, an antifungal agent, and
herbicides. Also, tubulin has been deeply involved in both the foundation
and application in life science.

[0009]A number of tubulin-polymerization inhibitors is hitherto known.
Vinca alkaloid is a presently well-known tubulin-polymerization inhibitor
used to treat cancers (Japanese Laid-Open Patent Application No.
5-192174). Vinca alkaloid is a generic term used for referring to one
kind of anticancer agents, the component of which is extracted from
plants. Vinca alkaloid binds to a protein called tubulin which is
involved in cell movement and cell shape maintenance, and inhibits
microtubules, whereby inhibiting cell division.

[0010]Representative examples of Vinca alkaloid include vinblastine and
vincristine (J. Med. Chem. 34:1998, 1991). Vinblastine is an indole
alkaloid having an antitumor activity among the alkaloids contained in
Catharanthus roseus, which acts on microtubules or tubulin which is a
component protein of microtubles, to stop cell mitosis in the metaphase
stage, thereby inhibiting cell division. Vincristine is now used for
treatment of leukemia, malignant lymphoma, pediatric tumor, etc. while
vinblastine is used for treatment of non-small cell cancer. In recent
years, vinblastine is prepared by a synthesis method (Japanese Laid-Open
Patent Application No. 2003-64084).

[0014]An object of the present invention is to provide a method for
introducing an extracellular substance into a mammalian oocyte, enabling
a safe and effective introduction into a mammalian oocyte when
introducing an extracellular substance such as a sperm, cell nucleus,
nucleic acid, and protein into a mammalian oocyte, which is technically
easy. A further object of the present invention is to provide a method
for introducing a sperm into a mammalian oocyte, which is technically
easy and causes less damage to the mammalian oocyte when introducing a
sperm into a mammalian oocyte.

Means to Solve the Object

[0015]The present inventors have conducted a keen study on a method for
introducing an extracellular substance such as a sperm into a mammalian
oocyte, and have found that, by treating a mammalian oocyte with a
tubulin-polymerization inhibitor such as vinblastine, a membrane fusion
between the oocyte and the sperm is promoted, thus fertilization can be
achieved by fusing

Patent Document 2: Japanese Laid-Open Patent Application No.

Patent Document 3: Japanese Laid-Open Patent Application No.

Patent Document 4: Japanese Laid-Open Patent Application No.

Patent Document 5: Japanese Laid-Open Patent Application No.

Patent Document 6: Japanese Laid-Open Patent Application No.

Patent Document 7: Japanese Laid-Open Patent Application No.

Patent Document 8: Japanese Laid-Open Patent Application No.

Patent Document 9: Japanese Laid-Open Patent Application

[0016]Patent Document 10: Published Japanese translation of PCT
international publication 8-503705Patent Document 11: Published Japanese
translation of PCT international publication 2001-500102Patent Document
12: Published Japanese translation of PCT international publication
2001-506225

[0018]More specifically, as a conventional method for introducing an
extracellular substance such as a sperm into a mammalian oocyte, the ICSI
method in which a hole is made in an oolemma with a glass capillary has
been used. The ICSI method is technically difficult and the success rate
thereof is low. Moreover, there is an adverse effect on embryonic
development caused by making a hole in the oolemma with a glass
capillary. Therefore, the present inventors have conducted a keen study
on a method for introducing a sperm which is technically easy and causes
less damages on the oocyte, and focused on development of a promoter for
introducing a sperm for a mammalian oocyte, which promotes membrane
fusion between an oocyte and a sperm.

[0019]Cytoplasmic membrane fusion is indispensable in order that a sperm
and oocyte undergo fusion to make a fertilized oocyte. Details regarding
a molecular mechanism for controlling membrane fusion during
fertilization are unknown, however, it has merged that microtubules
having tubulin as a constituent unit play an important roll from recent
studies of inventors. Given this factor, effects of
tubulin-polymerization promoter and inhibitor on membrane fusion during
fertilization was investigated, and it was found out that
tubulin-polymerization inhibitor, vinblastine, has a promotive effect on
the sperm fusion to an oolemma. Then, it was found out that this chemical
treatment enables sperm to be introduced into an oocyte without making a
hole in an oolemma.

[0020]That is, in order to investigate a mechanism in which vinblastine
treatment promotes membrane fusion, localization of membrane proteins in
a mouse oocyte which was fertilized in vitro after vinblastine treatment
was examined. The results revealed that localization of a
four-transmembrane protein CD9, which is believed to control membrane
fusion, was different from that of a wild type. The study hitherto have
revealed that it is essential that an area where CD9 is not present on an
oolemma is transiently formed by the sperm in order that membrane fusion
of fertilization occurs. It has been found that a circular area where CD9
is not present is formed before sperm binding by vinblastine treatment.
Furthermore, only one sperm fused to the center of a area where CD9 is
not present.

[0021]The present invention has been completed on the basis of findings
described above, and a method of the present invention can be applied not
only to the case of introducing a sperm into a mammalian oocyte, but also
to the case of introducing an extracellular substance such as a cell
nucleus, nucleic acid, and protein into a mammalian oocyte. That is, the
present invention relates to a method for introducing an extracellular
substance such as a sperm, cell nucleus, nucleic acid, and protein into a
mammalian oocyte by using a promoter for introducing an extracellular
substance for a mammalian oocyte containing a tubulin-polymerization
inhibitor as an active ingredient, and to an promoter for introducing an
extracellular substance for a mammalian oocyte for the same. Furthermore,
an aspect of the present invention is to provide a method for promoting
fertilization of a mammalian oocyte, by promoting introduction of a sperm
into a mammalian oocyte, by enhancing fertility of a mammalian oocyte by
a treatment with a promoter for introducing an extracellular substance
for a mammalian oocyte containing a tubulin-polymerization inhibitor as
an active ingredient.

[0022]Specifically, the present invention comprises: (1) a promoter for
introducing an extracellular substance for a mammalian oocyte used for
introducing an extracellular substance containing a
tubulin-polymerization inhibitor as an active ingredient into a mammalian
oocyte; (2) the promoter for introducing an extracellular substance for a
mammalian oocyte according to (1), wherein the tubulin-polymerization
inhibitor is vinblastine; (3) the promoter for introducing an
extracellular substance for a mammalian oocyte according to (1) or (2),
wherein the introduction of an extracellular substance into a mammalian
oocyte is the introduction of a sperm into the mammalian oocyte; (4) the
promoter for introducing an extracellular substance for a mammalian
oocyte according to (3), wherein the introduction of a sperm into the
mammalian oocyte is promoted by enhancement of fertility of the mammalian
oocyte; (5) the promoter for introducing an extracellular substance for a
mammalian oocyte according to any of (1) to (4), wherein the mammal is a
human; and (6) the promoter for introducing an extracellular substance
for a mammalian oocyte according to any of (1) to (4), wherein the mammal
is selected from the group consisting of a mouse, rat, cattle, pig,
horse, sheep, and monkey.

[0023]Moreover, the present invention comprises: (7) a method for
introducing an extracellular substance into a mammalian oocyte, wherein
the mammalian oocyte is treated with a promoter for introducing an
extracellular substance for a mammalian oocyte containing a
tubulin-polymerization inhibitor as an active ingredient, when
introducing the extracellular substance into the mammalian oocyte; (8)
the method for introducing an extracellular substance into a mammalian
oocyte according to (7), wherein the mammalian oocyte is treated with
vinblastine, when introducing the extracellular substance into the
mammalian oocyte; (9) the method for introducing an extracellular
substance into a mammalian oocyte according to (7) or (8), wherein the
introduction of an extracellular substance into a mammalian oocyte is the
introduction of a sperm into a mammalian oocyte; (10) the method for
introducing an extracellular substance into a mammalian oocyte according
to (9), wherein the introduction of an extracellular substance into a
mammalian oocyte is achieved by sperm-oocyte fusion outside a mammalian
body; (11) the method for introducing an extracellular substance into a
mammalian oocyte according to (9) or (10), wherein the introduction of
the sperm into a mammalian oocyte is promoted by improvement of fertility
of the mammalian oocyte; and (12) the method for introducing an
extracellular substance into a mammalian oocyte according to (11),
wherein improvement of the fertility of the mammalian oocyte is promotion
of sperm fusion to an oolemma.

BRIEF DESCRIPTION OF FIGURES

[0024]FIG. 1 It is a figure showing the result of the number of sperm
fused with an oocyte as a result of in vitro fertilization of mouse sperm
and a zona pellucida-free mouse unfertilized oocyte which has been
treated with vinblastine, a tubulin-polymerization inhibitor, in an
example of the present invention.

[0025]FIG. 2 It is a figure showing the result of the ratio of fertilized
oocytes resulted from in vitro fertilization of mouse sperm and an
unfertilized mouse oocyte with zona pellucida which has been treated with
vinblastine, a tubulin-polymerization inhibitor, in an example of the
present invention.

BEST MODE OF CARRYING OUT THE INVENTION

[0026]A method of the present invention includes treating the mammalian
oocyte with a promoter for introducing an extracellular substance for the
mammalian oocyte containing a tubulin-polymerization inhibitor as an
active ingredient, and introducing the extracellular substance into the
mammalian oocyte when introducing an extracellular substance such as
sperm into a mammalian oocyte. As a tubulin-polymerization inhibitor used
as an active ingredient of the promoter for introducing an extracellular
substance for the mammalian oocyte of the present invention, a
tubulin-polymerization inhibitor which is publicly known as an inhibitor
for tubulin polymerization, can be used. The tubulin-polymerization
inhibitor includes vinca alkaloid, and typical examples of vinca alkaloid
include vinblastine and vincristine. As a most preferable active
ingredient of the promoter for introducing an extracellular substance for
the mammalian oocyte of the present invention, vinblastine can be
exemplified.

[0027]A mammalian oocyte of interest in the present invention includes,
but not especially limited to, an oocyte of a human, mouse, rat, cattle,
pig, horse, sheep, and monkey. A method of introducing an extracellular
substance into a mammalian oocyte according to the present invention can
be applied to extracellular substances such as sperm, a cell nucleus, a
nucleic acid, and a protein. However, the method for introducing an
extracellular substance according to the present invention can be
especially applied to the introduction of a sperm into a mammalian
oocyte, thus can be provided as an effective method of fertilization of a
mammalian oocyte.

[0028]In the present invention, the treatment of a mammalian oocyte with
the promoter for introducing an extracellular substance for the mammalian
oocyte containing a tubulin-polymerization inhibitor as an active, can be
carried out by: collecting an oocyte, culturing it in an adequate medium
(e.g. TYH medium) followed by culturing it in the medium added with a
tubulin-polymerization inhibitor for a few hours.

[0029]Although the invention will be described in more detail in the
following by way of examples, the technical scope of the present
invention is not limited thereto.

EXAMPLE 1

Increase in Fertilization Efficiency by Vinblastine Treatment

[0030]In order to examine the influence of a tubulin-polymerization
inhibitor on fertilization of a mouse unfertilized oocyte, vinblastine
was used as a tubulin-polymerization inhibitor to treat a mouse
unfertilized oocyte. Then the fertility thereof was investigated.

[0032](1) Mice of eight week-old or older were purchased and made
adapted to grow environment for 1 week before use. PMS (Serotoropin
exclusive to animal, manufactured by ASUKA Pharmaceutical) was injected
in an amount of 0.1 ml into the abdominal cavity of the mouse at 8:00 pm.
[0033](2) 48 hr later, HCG (Gonadotropin exclusive to animal,
manufactured by ASUKA Pharmaceutical) was injected in an amount of 0.1 ml
into the abdominal cavity. [0034](3) Mice were euthanized the next day
(14 to 16 hr after HCG injection), and the ampulla of oviduct was
removed. An oocyte surrounded by cumulus cells was collected and cultured
in a (100 μL) drop of TYH medium. [0035](4) Cumulus cells were removed
by hyaluronidase (300 μg/mL) treatment, then the oocyte was cultured
in TYH medium for 3 hr. In the case of conducting removal of zona
pellucida, oocyte zona pellucida was removed by an acid Tyrode's solution
treatment followed by culturing the oocyte in TYH medium for 3 hr.2.
Treatment with a Tubulin-Polymerization Inhibitor

[0036]The collected unfertilized oocyte of the mice was treated by using
vinblastine (VB) as a tubulin-polymerization inhibitor. As controls, an
oocyte treated with paclitaxel (Pac; Brand Name: taxol),
tubulin-polymerization promoter, and an untreated oocyte (control) were
used. Vinblastine (VB) treatment was conducted by culturing the oocyte in
20 μM of vinblastine solution (TYH medium) for 1 hr, while paclitaxel
(Pac) treatment was conducted by culturing the oocyte in 10 μM of
paclitaxelel (Pac) solution (TYH medium) similarly for 1 hr.

3. In Vitro Fertilization

[0037]The mouse unfertilized oocyte was treated with vinblastine (VB), a
tubulin-polymerization inhibitor, and in vitro fertilization was
conducted with mouse sperm. As controls, an unfertilized oocyte treated
with paclitaxel (Pac; Brand Name: taxol), tubulin-polymerization
promoter, and an untreated oocyte (control) were used.

(Results)

[0038](In the case where zona pellucida-free oocytes were use): To
quantify sperm-oocyte fusion efficiency, zona pellucida-free mouse
unfertilized oocytes were treated with vinblastine (VB), a
tubulin-polymerization inhibitor, or paclitaxel (Pac),
tubulin-polymerization promoter, or left untreated (control). Then the
oocytes were in vitro fertilized with mouse sperm, respectively. The
results are shown in FIG. 1. The figure shows the results of in vitro
fertilization (zona pellucida-free oocytes, 1 hr after insemination). In
the figure, white bars show the number of fused sperm. When comparing the
number of sperm which fused with the oocytes 1 hr after insemination, the
VB (vinblastine)-treated oocyte showed the largest number. On the other
hand, a large number of sperm stuck to the surface of the paclitaxel
(Pac)-treated oocyte but not fused therewith, because the paclitaxel
(Pac)-treated oocyte made difficult sperm to be fused.

[0039](In the case where oocytes with zona pellucida are used): Mouse
unfertilized oocytes just after ovulation, which had not been undergone
special treatment such as removal of zona pellucida, were treated with
vinblastine, a tubulin-polymerization inhibitor, and in vitro fertilized
with mouse sperm. The results are shown in FIG. 2. An experiment using
anti-mouse CD9 antibody having a fusion-inhibitory effect was conducted
simultaneously with this experiment. In the case of mouse, about 20% of
oocytes cannot be fertilized due to immaturity and over-maturity.
However, after investigating the fertilization efficiency using the
2-cell-stage oocytes, the fertilization efficiency of the
vinblastine-treated oocytes increased by 20% comparing to that of the
untreated oocyte. As it will be described in the followings, even an
immature oocyte could be fused with sperm when treated with vinblastine.
So, it is believed that this is the result of the increased fusion
efficiency of sperm-oocyte fusion due to the treatment with vinblastine.
The experimental result reveals that either immature or over-mature
oocytes become possible to fuse with sperm by vinblastine treatment.
Furthermore, fusion efficiency of oocytes which have been treated with
anti-mouse CD9 antibody having a fusion-inhibitory effect, was also
increased by vinblastine treatment.

(Increase of Fertilization Efficiency by Vinblastine Treatment)

[0040]After treating mouse unfertilized oocytes with vinblastine, a
tubulin-polymerization inhibitor, the oocytes were in vitro fertilized
with mouse sperm. The results revealed that fertilization efficiency
thereof increased by 20% comparing to untreated group. In the case of
mouse, about 20% of oocytes cannot be fused with sperm due to immaturity.
However, even immature oocytes could fuse with sperm when treated with
vinblastine.

(Treatment with Paclitaxelel)

[0041]When a mouse unfertilized oocyte was treated with paclitaxelel
(Pac), tubulin-polymerization promoter, sperm-oocyte membrane fusion was
inhibited. A number of deformed microvilli were observed when the
morphology of oocyte membrane was examined with an electron microscope.

(Formation of Sperm Fusion Area by Vinblastine Treatment)

[0042]The mechanism in which membrane fusion was promoted by vinblastine
treatment was investigated. After vinblastine treatment, the localization
of membrane proteins in an in vitro fertilized mouse oocyte was examined.
When treated with vinblastine, a tubulin-polymerization inhibitor,
circular areas where localization of CD9 on the oocyte membrane does not
change were formed. The number of circular areas differed depending on
oocytes and were ranging from 1 to 6. Moreover, it was confirmed that one
sperm was fused to the center of circular areas. This shows that tubulin
depolymerization is necessary for sperm fusion.

[0043]Namely, the results revealed that the localization of a
four-transmembrane protein CD9, which is believed to control membrane
fusion, was different from that of a wild type. The study hitherto has
revealed that it is essential that an area where CD9 is not present is
transiently formed by the sperm on an oolemma for the occurrence of
membrane fusion of fertilization. It has been found that a circular area
where CD9 is not present is formed by vinblastine treatment before sperm
binding. Furthermore, only one sperm fused to the center of an area where
CD9 is not present. In addition, it was confirmed that, in fertilization
of an oocyte with a semipermeable membrane, the number of sperm to be
introduced into the oocyte is limited to one due to its polyspermy block
mechanism. From these results, it has became apparent that, by treating
the specific area of oocyte membrane with vinblastine, the introduction
of sperm into an oocyte becomes possible without using a glass capillary.

INDUSTRIAL APPLICABILITY

[0044]The present invention can provide a method for introducing an
extracellular substance into a mammalian oocyte, enabling a safe and
effective introduction into a mammalian oocyte when introducing an
extracellular substance such as a sperm, cell nucleus, nucleic acid, and
protein into a mammalian oocyte, which is technically easy. Specifically,
a method of the present invention can be applied as an introduction
method of a sperm into a mammalian oocyte, and provide a technically easy
method without using a special device such as that used in the
conventional ICSI method, and enables sperm to be introduced into a
mammalian oocyte causing less damages on the mammalian oocyte, because it
is not necessary to make a hole in the oolemma with a glass capillary as
in the ICSI method. Therefore, it can be expected that the present
invention can contribute to the development in the field of mammalian
modification using gene therapy, reproductive treatment, or developmental
engineering, as a useful means for introducing an extracellular substance
such as a sperm, cell nucleus, nucleic acid, and protein, in a mammalian
oocyte, for fertilization or modification of germ cells.